Structural basis of actin sequestration by thymosin-beta4: implications for WH2 proteins

Abstract

The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-beta4 (Tbeta4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tbeta4 (G1-Tbeta4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tbeta4:actin complex at 2 A resolution. The structure reveals that Tbeta4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tbeta4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motif-containing proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.

Figure 1

Characterization of the effect of G1+ and G1-Tβ4 on actin filaments. (A, B) Time course of actin polymerization in the presence of G1+ or G1-Tβ4. The fluorescence intensity of 4 μM actin (5% pyrene labeled) was followed during polymerization. (A) Black, actin alone; red, addition of 20 nM G1+; blue, 270 nM G1+; and green, 540 nM G1+. (B) Black, actin alone; red, addition of 50 nM G1-Tβ4; blue, 200 nM G1-Tβ4; and green, 600 nM G1-Tβ4. (C) Elongation assay for the effect of G1+ or G1-Tβ4 on the number of free actin filament ends. Mixtures of G1+ or G1-Tβ4 and 1 μM actin were equilibrated before the addition of 1 μM pyrene–actin monomers and polymerization was followed by fluorescence. The dependence of the relative initial rate of polymerization was calculated from the initial slope of the elongation curves and is plotted against concentration of G1+ (black) and G1-Tβ4 (red). (D–F) Fluorescence micrographs of (D) actin filaments, (E) actin filaments incubated with 80 nM G1+ and (F) actin filaments incubated with 80 nM G1-Tβ4.

Figure 2

Tβ4 interactions with G-actin. (A) Structure of the G1-Tβ4:actin complex. The actin protomer is shown in sky blue with a bound ATP in orange (ball-and-stick representation) and a calcium ion (green sphere). The actin subdomains are labeled 1–4. The G1 portion of the hybrid (residues 27–149) is shown in royal blue with two associated calcium ions (dark spheres). The Tβ4 portion (residues 21–39) is depicted in gold. The red region of the G1-Tβ4 ribbon represents the G1 sequence that is homologous to the Tβ4 sequence (residues 17–20 in Tβ4). (B) A 90° rotation (clockwise when viewed from above) around the vertical axis compared to (A). (C) Model of Tβ4 bound to actin. Actin and Tβ4 residues 17–39 are taken from the structure in (B). The Tβ4 N-terminus (pink) is modeled by taking the homologous amino acids from the ciboulot:actin structure (PDB code 1SQK; Hertzog et al, 2004) after superimposing the actin structures. (D) Stereo view of the structural overlap within the LKKTET-related motif between the actin-bound forms of G1-Tβ4 and ciboulot. The actin structures from the present structure and the ciboulot:actin structure (PDB code 1SQK; Hertzog et al, 2004) were superimposed. Gelsolin (G) residues Phe149-Lys150-His151-Val152 and Tβ4 (T) residue Glu21 from the hybrid are shown in green and are labeled in the left panel. The homologous ciboulot (C) residues Leu30-Lys31-Asn32-Ala33-Ser34 are shown in pink and are labeled in the right panel.

Figure 3

Conformational changes in the β-thymosin family on binding actin. (A) Stereo view of a representative portion of the 2F_o–F_c electron density covering Tβ4 residues 23–39, contoured at 1.2σ. The orientation is similar to that in Figure 2B. (B) The solution structure of Tβ9 (PDB code 1HJ0; Stoll et al, 1997). (C) Solution structure of Tβ4 (Czisch et al, 1993). (D) Model of actin-bound Tβ4 from Figure 2C. In each representation, the models are aligned based on the C-terminal minus-end capping helix and colored as in Figure 2B.

Figure 4

Sequence alignment of the WH2 family of proteins based on structural considerations. Yellow, conserved residues in the Tβ4 structure; pink, conserved residues in the G1 structure; gray, Tβ4 homologous residues in the model; pale blue, conserved residues in the model throughout the WH2 family with the exception of the Tβ4 subfamily; orange, acidic residues within the A region; green, nonacidic conserved residues in the CA regions that are not related to the WH2 family. The CA regions are shown boxed. The C regions appear to have homology with the WH2 motif. The A regions are included for size comparison and show no homology with the WH2 family. All sequences are human, except Tβ9 (bovine), actobindin (amoeba) and ciboulot (Drosophila).

Figure 5

Competition for the Tβ4 actin-binding site. (A) Model of the competition between Tβ4 and DNase I for binding actin. The structure of actin:DNase I (PDB code 1ATN; Kabsch et al, 1990) and the Tβ4:actin model are superimposed, with only one actin shown. DNase I is drawn in red. The arrow indicates a structural clash. (B) Model of the competition between Tβ4 and profilin for binding actin. The structure of profilin:actin (PDB code 2BTF; Schutt et al, 1993) has been superimposed on the Tβ4:actin model, and profilin is depicted in red. The arrow indicates a structural clash. (C) Model of the interactions of the WH2 domain family with F-actin. Tβ4 docked onto the side of an actin filament based on superimposing the actins from two copies of the Tβ4:actin model (Figure 2C) on two actins from a modified version of the Holmes model of the filament (Holmes et al, 1990). The actins are colored sky blue and Tβ4s are painted as in Figure 2A. This representation shows that capping by the N-terminal helix and the minus-end capping helix prevent the Tβ4:actin complex from joining either end of a filament. The model also demonstrates that multiple WH2 repeat proteins will bind actin protomers in a longitudinal manner, along the axis of the filament.

Figure 6

Model of the role of the CA motif in arp2/3 activation. Tβ4 docked onto arp2 within the ATP model of arp2/3 (based on PDB code 1K8K; Robinson et al, 2001). Arp2/3 subunits are colored as follows: Arp3, red; arp2, yellow; ARC1, green; ARC2, royal blue; ARC3, leaf green; ARC4, cyan; and ARC5, purple. The Tβ4:actin model is superimposed on arp2, with only Tβ4 (black) retained in the figure with its N-terminus labeled N. In this figure, Tβ4 residues 1–25 represent the C region of the VCA arp2/3 activators, and residues 26–39 indicate the size and approximate location of the A region. This model is not meant to represent the true structure of the A region, rather to demonstrate that if the A region were to adopt an extended conformation, then it would be of an appropriate size and in the right locale to be able to contact both arp3 (red) and ARC3 (leaf green).